Automation

Extract-N-Amp Plant Kit


Large-scale plant genomic and breeding studies have created the need for automated methodologies for genomic DNA preparation. The Extract-N-Amp™ Plant PCR kit is a high-throughput system for the rapid extraction and subsequent amplification of genomic DNA from plant leaves (Figures 1, 2, 3 and 4). Scientists at Sigma-Aldrich have developed a novel extraction treatment that releases genomic DNA from leaf punches in minutes. Without further cleanup, the DNA is ready for direct use in PCR applications. To further simplify the procedure the Extract-N-Amp Plant PCR kit includes a specially formulated PCR ReadyMix™, which virtually eliminates any PCR optimization.

Extract-N-Amp Plant PCR Kit, containing Sigma Advanced Technology, offers an ideal platform for automated genomic screening of plant leaf samples. Features of the procedure include the following:

  • The entire process from leaf punches through PCR reaction setup is fully automated.
  • Rapid method – 96 leaf samples can be processed in 30 minutes.
  • No upfront preparation of leaf tissue required such as freezing or mechanical disruption.

 



Automation Resources

Automated methods have been developed and validated for the Extract-N-Amp Plant PCR Kit. The following resources are available for download:



Applications

Automation of Extract-N-Amp Plant PCR Kit is suitable for a variety of applications including:

  • Marker-assisted breeding
  • Genotyping of transgenic plants
  • Genomic mapping

 


Features & Benefits

Walk-Away Automation A liquid handler, such as the Sciclone ALH 3000 Workstation, automates all aspects of the Extract-N-Amp Plant Kit including extraction of genomic DNA and PCR reaction setup.
Rapid Procedure 96 leaf punches can be extracted and setup for PCR in 30 minutes. There is no need for freezing or mechanical disruption.
Enhanced Productivity Walk-away automation of genomic DNA extraction and PCR reaction setup allows you to concentrate on other aspects of your work.
Flexible Compatible with a variety of plant types including Maize, soybean, tobacco, and tomato.
Convenient Kits are packaged with a PCR reaction mix, optimally formulated for use with leaf extracts.

REDExtract-N-Amp ReadyMix contains an inert tracking dye to allow for direct loading of PCR reactions onto an agarose gel.

The SYBR Green Extract-N-Amp PCR ReadyMix allows for real-time quantitative analysis of the amplified PCR products.
High Specificity PCR ReadyMix includes an antibody-mediated Hot Start mechanism for highly specific amplification.
Stable Extracts Once extracts are neutralized, they are stable for at least 6 months if stored at 4° C.


Extract-N-Amp Plant PCR Kit  
Product #  Product Name Number of Extractions Number of Amplifications
XNAR Extract-N-Amp Plant PCR 1000 1000
REDExtract-N-Amp Plant PCR Kit  
Product #  Product Name Number of Extractions Number of Amplifications
XNAPR REDExtract-N-Amp Plant PCR 1000 1000

Kit Contents

  • Extraction Solution
  • Dilution Solution
  • REDExtract-N-Amp or Extract-N-Amp PCR ReadyMix

 

Product #  Product Name Number of Amplifications
E 3004 Extract-N-Amp PCR ReadyMix 1000
R 4775 REDExtract-N-Amp PCR ReadyMix 1000


PCR analysis of genomic DNA isolated from 88 different corn leaf punches using an automated method for the Extract-N-Amp Plant PCR Kit.

Figure 1. Agarose gel analysis of 96 PCR samples.

DNA was extracted from 88 different corn leaves (0.5–0.7 cm disks) using the automated Extract-N-Amp Plant PCR procedure on the Sciclone ALH 3000. Amplification of 438 bp fragment of the universal chloroplast genomic DNA followed using 4 µl of extracted template or 4 µl of corn genomic DNA controls (A12, C12, E12, and G12) in a 20 µl PCR reaction incorporating the 2x PCR ReadyMix. 6µl of each reaction was analyzed on a 2% agarose gel. Lanes B12, D12, F12, and H12 represent no template controls.



Cross Contamination Analysis of the Automated Extract-N-Amp Plant Method.

Figure 2. Agarose gel analysis of cross-contamination test.

Corn leaf punches (0.5–0.7 cm disks) were placed in alternating wells of a 96 well plate. The plate was processed using the automated Extract-N-Amp™ Plant PCR procedure on the Sciclone ALH 3000. All samples were then subjected to amplification and 6 µl of the resultant products were electrophoresed on a 2% agarose gel. No PCR products were detected in the wells containing no plant tissue samples.



PCR analysis of genomic DNA isolated from various plant types using an automated method for the Extract-N-Amp Plant PCR Kit.

Figure 3. Agarose gel analysis of PCR samples from different plant species.

DNA was extracted from soybean, tobacco, tomato, and corn leaves using the automated Extract-N-Amp™ Plant PCR procedure on the Sciclone ALH 3000. Amplification of 400–500 bp fragment of the universal chloroplast gene followed using 4 µl of extracted template DNA or 4 µl of corn genomic DNA controls (Lanes A12 and C12) in a 20 µl PCR reaction incorporating the 2x PCR ReadyMix. 6 µl of each reaction was loaded on a 2% agarose gel, with soybean samples in lanes A1–A11, tobacco samples in lanes B1–B11, tomato samples in lanes C1–C11, and corn samples in lanes D1–D11.



Quantitative PCR Analysis

Figure 4.

Eighty-eight tomato leaf samples were extracted using the SYBR Green Extract-N-Amp Plant PCR Kit following the automated procedures. Reaction analyses were performed on an ABI Prism 7700 Sequence Detection System. The graph was plotted as the intensity of florescence in logarithms versus the value of cycle threshold (CT).


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